Interleukin-1 Alpha Antibodies and Methods of Use

ABSTRACT

Fully human monoclonal Abs includes (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1α and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is continuation application of U.S.nonprovisional patent application Ser. No. 13/224,913, filed on Sep. 2,2011, which is a divisional application of U.S. nonprovisional patentapplication Ser. No. 12/455,458, filed on Jun. 1, 2009 (now U.S. Pat.No. 8,034,337), which claims the priority of U.S. provisional patentapplication Ser. Nos. 61/057,586; 61/121,391; and 61/178,350 filed onMay 30, 2008; Dec. 10, 2008; and May 14, 2009, respectively.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention relates generally to the fields of immunology,inflammation, cancer, vascular disorders, and medicine. Moreparticularly, the invention relates to antibodies (Abs) whichspecifically bind interleukin-1α (IL-1α) and methods of using such Absto treat, prevent, or detect a pathology associated with aberrant IL-1αexpression.

BACKGROUND

IL-1α is pro-inflammatory cytokine that plays a role in a number ofdifferent activities including inflammation, immune responses, tumormetastasis, and hematopoiesis. IgG autoantibodies against IL-1α occurnaturally in the general human population and are thought to bebeneficial in diseases such as atherosclerosis.

SUMMARY

The invention is based on the development of fully human monoclonal Abs(mAbs) that include (i) an antigen-binding variable region that exhibitsvery high binding affinity for human IL-1α and (ii) a constant regionthat is effective at both activating the complement system though C1qbinding and binding to several different Fc receptors. The IL-1αspecific mAbs described herein was made by replacing the constant regionof a human IgG4 mAb having a variable region specific for human IL-1αwith the constant region of a human IgG1 mAb.

Accordingly, the invention features a purified human IgG1 mAb thatspecifically binds to human IL-1α, the mAb including a heavy chaincovalently joined to a light chain. The heavy chain can include theamino acid sequence of SEQ ID NO: 9 and the light chain can include theamino acid sequence of SEQ ID NO:11.

Also within the invention is a set of isolated nucleic acids including afirst nucleic acid encoding the heavy chain of a human IgG1 mAb thatspecifically binds to IL-1α, and a second nucleic acid encoding thelight chain of the human IgG1 mAb that specifically binds to humanIL-1α. The first nucleic acid can encode the amino acid sequence of SEQID NO: 9 and the second nucleic acid can encode the amino acid sequenceof SEQ ID NO:11. The first nucleic acid can include the nucleotidesequence of SEQ ID NO: 10 and the second nucleic acid can include thenucleotide sequence of SEQ ID NO:12.

In another aspect, the invention features an expression vector includinga nucleic acid encoding the amino acid sequence of SEQ ID NO: 9 or SEQID NO: 11.

Another feature of the invention is an isolated host cell (e.g. amammalian cell such as a CHO cell) including set of isolated nucleicacids including a first nucleic acid encoding the heavy chain of a humanIgG1 mAb that specifically binds to IL-1α, and a second nucleic acidencoding the light chain of the human IgG1 mAb that specifically bindsto human IL-1α. The heavy chain can include the amino acid sequence ofSEQ ID NO: 9 and a light chain can include the amino acid sequence ofSEQ ID NO:11.

The invention further features a method of killing a cell expressinghuman IL-1α. This method can include the step of contacting the cellwith a purified human IgG1 mAb that specifically binds to human IL-1α.

A method of inhibiting migration of a human cell through a basementmembrane matrix is also within the invention. This method can includethe step of adding a purified mAb that specifically binds to human IL-1αto a mixture including a basement membrane matrix and the human cell.

Further within the invention is a method of inhibiting an IL-1α-inducedincrease in ICAM-1 and/or E-selectin expression on the surface of ahuman endothelial cell. This method can include the step of adding apurified mAb that specifically binds to human IL-1α to a mixtureincluding the endothelial cell and IL-1α.

The invention additionally includes a method of tracking inflammation ina human subject previously subjected to the steps of: obtaining from thesubject a first sample of peripheral blood mononuclear cells at a firsttime; contacting the first sample with a purified mAb that specificallybinds to human IL-1α; and determining the percent of cells in the firstsample that bind the monoclonal Ab. This method can include the stepsof: (a) obtaining from the subject a second sample of peripheral bloodmononuclear cells at a second time; (b) contacting the second samplewith the purified mAb that specifically binds to human IL-1α; (c)determining the percent of cells in the second sample that bind themonoclonal Ab; and (d) comparing the percent of cells in the firstsample that bind the mAb to the percent of cells in the second samplethat bind the monoclonal Ab.

In the foregoing methods, the purified mAb can be a human IgG1 mAbincluding a heavy chain covalently joined to a light chain, e.g.,wherein the heavy chain includes the amino acid sequence of SEQ ID NO: 9and the light chain includes the amino acid sequence of SEQ ID NO:11.

Another method within the invention features the steps of: (a) enrichinga biological sample obtained from a human subject using a filter toseparate molecules according to molecular weight into a first fractionincluding intact IgG complexed with IL-1α and second fraction includingmolecules less than 100 Kda; and (b) quantifying the amount of IL-1α inthe first fraction.

Yet another method within the invention features the steps of: (a)enriching a sample of plasma obtained from a human subject using afilter that separates molecules according to molecular weight into afirst fraction including intact IgG complexed with IL-1α and secondfraction including molecule less than 100 Kda; (b) adding the firstfraction to a substrate including immobilized anti-human IgG Abs underconditions that allow IgG in the first fraction to specifically bind theanti-human IgG Abs immobilized on the substrate; (c) washing thesubstrate to remove material in the first fraction that does notspecifically bind the immobilized anti-human IgG Abs; (d) contacting thesubstrate washed in step (c) with an Ab that specifically binds humanIL-1α under conditions that allows the Ab that specifically binds humanIL-1α to specifically bind any human IL-1α bound to the substrate; (e)washing the substrate to remove any of the Ab that specifically bindshuman IL-1α that is not bound to the substrate; and (f) quantifying theamount of Ab that specifically binds human IL-1α remaining bound to thesubstrate after step (e).

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofbiological terms can be found in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994.

The term “specifically binds”, as used herein, when referring to apolypeptide (including Abs) or receptor, refers to a binding reactionwhich is determinative of the presence of the protein or polypeptide orreceptor in a heterogeneous population of proteins and other biologics.Thus, under designated conditions (e.g. immunoassay conditions in thecase of an Ab), the specified ligand or Ab binds to its particular“target” and does not bind in a significant amount to other proteinspresent in the sample or to other proteins to which the ligand or Ab maycome in contact in an organism. Generally, a first molecule that“specifically binds” a second molecule has an equilibrium affinityconstant greater than about 10⁵ (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,and 10¹² or more) liters/mole for that second molecule.

When referring to a protein molecule such as an Ab, “purified” meansseparated from components that naturally accompany such molecules.Typically, an Ab or protein is purified when it is at least about 10%(e.g., 9%, 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.9%, and 100%), by weight, free from the non-Ab proteins or othernaturally-occurring organic molecules with which it is naturallyassociated. Purity can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. A chemically-synthesized protein or other recombinant proteinproduced in a cell type other than the cell type in which it naturallyoccurs is “purified.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All applications andpublications mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions will control. In addition, the particular embodimentsdiscussed below are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION

The invention encompasses compositions and methods relating to fullyhuman mAbs that include (i) an antigen-binding variable region thatexhibits very high binding affinity for IL-1α and (ii) a constant regionthat is effective at both activating the complement system though C1qbinding and binding to several different Fc receptors. The belowdescribed preferred embodiments illustrate adaptation of thesecompositions and methods. Nonetheless, from the description of theseembodiments, other aspects of the invention can be made and/or practicedbased on the description provided below.

Methods involving conventional immunological and molecular biologicaltechniques are described herein. Immunological methods (for example,assays for detection and localization of antigen-Ab complexes,immunoprecipitation, immunoblotting, and the like) are generally knownin the art and described in methodology treatises such as CurrentProtocols in Immunology, Coligan et al., ed., John Wiley & Sons, NewYork. Techniques of molecular biology are described in detail intreatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology,Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York.Ab methods are described in Handbook of Therapeutic Abs, Dubel, S., ed.,Wiley-VCH, 2007. Cell culture techniques are generally known in the artand are described in detail in methodology treatises such as Culture ofAnimal Cells: A Manual of Basic Technique, 4th edition, by R IanFreshney, Wiley-Liss, Hoboken, N.J., 2000; and General Techniques ofCell Culture, by Maureen A Harrison and Ian F Rae, Cambridge UniversityPress, Cambridge, UK, 1994. Methods of protein purification arediscussed in Guide to Protein Purification: Methods in Enzymology, Vol.182, Deutscher M P, ed., Academic Press, San Diego, Calif., 1990.

In one aspect, the invention features a fully human mAb that includes(i) an antigen-binding variable region that exhibits very high bindingaffinity for human IL-1α and (ii) a constant region that is effective atboth activating the complement system though C1q binding and binding toseveral different Fc receptors. The human Ab is preferably an IgG1. TheKa of the Ab is preferably at least 1×10⁹ M⁻¹ or greater (e.g., greaterthan 9×10¹⁰ M⁻¹, 8×10¹⁰ M⁻¹, 7×10¹⁰ M⁻¹, 6×10¹⁰ M⁻¹, 5×10¹⁰ M⁻¹, 4×10¹⁰M⁻¹, 3×10¹⁰ M⁻, 2×10¹⁰ M⁻¹, or 1×10¹⁰ M⁻¹).

Because B lymphocytes which express Ig specific for human IL-1α occurnaturally in human beings, a presently preferred method for raising mAbsis to first isolate such a B lymphocyte from a subject and thenimmortalize it so that it can be continuously replicated in culture.Subjects lacking large numbers of naturally occurring B lymphocyteswhich express Ig specific for human IL-1α may be immunized with one ormore human IL-1α antigens to increase the number of such B lymphocytes.Human mAbs are prepared by immortalizing a human Ab secreting cell(e.g., a human plasma cell). See, e.g., U.S. Pat. No. 4,634,664.

In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, ormore) human subjects (e.g., subjects not previously administered a humanIL-1α vaccine) are screened for the presence of such humanIL-1α-specific Ab in their blood. Those subjects that express thedesired Ab can then be used as B lymphocyte donors. In one possiblemethod, peripheral blood is obtained from a human donor that possesses Blymphocytes that express human IL-1α-specific Ab. Such B lymphocytes arethen isolated from the blood sample, e.g., by cells sorting (e.g.,fluorescence activated cell sorting, “FACS”; or magnetic bead cellsorting) to select B lymphocytes expressing human IL-1α-specific Ig.These cells can then be immortalized by viral transformation (e.g.,using EBV) or by fusion to another immortalized cell such as a humanmyeloma according to known techniques. The B lymphocytes within thispopulation that express Ig specific for human IL-1α can then be isolatedby limiting dilution methods (e.g., cells in wells of a microtiter platethat are positive for Ig specific for human IL-1α are selected andsubcultured, and the process repeated until a desired clonal line can beisolated). See, e.g., Goding, Monoclonal Abs: Principles and Practice,pp. 59-103, Academic Press, 1986. Those clonal cell lines that expressIg having at least nanomolar or picomolar binding affinities for humanIL-1α are preferred. MAbs secreted by these clonal cell lines can bepurified from the culture medium or a bodily fluid (e.g., ascites) byconventional Ig purification procedures such as salt cuts, sizeexclusion, ion exchange separation, and affinity chromatography.

Although immortalized B lymphocytes might be used in in vitro culturesto directly produce mAbs, in certain cases it might be desirable to useheterologous expression systems to produce mAbs. See, e.g., the methodsdescribed in U.S. patent application Ser. No. 11/754,899. For example,the genes encoding an mAb specific for human IL-1α might be cloned andintroduced into an expression vector (e.g., a plasmid-based expressionvector) for expression in a heterologous host cell (e.g., CHO cells, COScells, myeloma cells, and E. coli cells). Because Igs include heavy (H)and light (L) chains in an H₂L₂ configuration, the genes encoding eachmay be separately isolated and expressed in different vectors.

Although generally less preferred, chimeric mAbs (e.g., “humanized”mAbs), which are antigen-binding molecules having different portionsderived from different animal species (e.g., variable region of a mouseIg fused to the constant region of a human Ig), might be used in theinvention. Such chimeric Abs can be prepared by methods known in theart. E. G., Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984. Similarly, Abs can be humanized by methods known in the art. Forexample, monoclonal Abs with a desired binding specificity can becommercially humanized or as described in U.S. Pat. Nos. 5,693,762;5,530,101; or U.S. Pat. No. 5,585,089.

The mAbs described herein might be affinity matured to enhance orotherwise alter their binding specificity by known methods such as VHand VL domain shuffling (Marks et al. Bio/Technology 10:779-783, 1992),random mutagenesis of the hypervariable regions (HVRs) and/or frameworkresidues (Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994;Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol.155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995;and Hawkins et al, J. Mol. Biol. 226:889-896, 1992. Amino acid sequencevariants of an Ab may be prepared by introducing appropriate changesinto the nucleotide sequence encoding the Ab. In addition, modificationsto nucleic acid sequences encoding mAbs might be altered (e.g., withoutchanging the amino acid sequence of the mAb) for enhancing production ofthe mAb in certain expression systems (e.g., intron elimination and/orcodon optimization for a given expression system). The mAbs describedherein can also be modified by conjugation to another protein (e.g.,another mAb) or non-protein molecule. For example, a mAb might beconjugated to a water soluble polymer such as polyethylene glycol or acarbon nanotube (See, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See, U.S. patent application Ser. No. 11/754,899.

Preferably, to ensure that high titers of human IL-1α-specific mAb canbe administered to a subject with minimal adverse effects, the mAbcompositions of the invention are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,95, 96, 97, 98, 99, 99.9 or more percent by weight pure (excluding anyexcipients). The mAb compositions of the invention might include only asingle type of mAb (i.e., one produced from a single clonal B lymphocyteline) or might include a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10 or more) different types of mAbs. In addition to human IL-1αmAbs, the Ab compositions of the invention might also include other mAbsthat specifically bind antigens other than human IL-1α.

To modify or enhance their function, the human IL-1α mAbs might beconjugated another molecule such as a cytotoxin or detectable label. Ahuman IL-1α specific mAb might be conjugated with one or more cytotoxinsto more effectively kill cells expressing IL-1α. Cytotoxins for use inthe invention can be any cytotoxic agent (e.g., molecule that can kill acell after contacting the cell) that can be conjugated to a human IL-1αspecific mAb. Examples of cytotoxins include, without limitation,radionuclides (e.g., ³⁵S, ¹⁴C, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁹Zr, ²⁰¹Tl, ¹⁸⁶Re,¹⁸⁸Re, ⁵⁷Cu, ²¹³Bi, and ²¹¹At), conjugated radionuclides, andchemotherapeutic agents. Further examples of cytotoxins include, but arenot limited to, antimetabolites (e.g., 5-fluorouricil (5-FU),methotrexate (MTX), fludarabine, etc.), anti-microtubule agents (e.g.,vincristine, vinblastine, colchicine, taxanes (such as paclitaxel anddocetaxel), etc.), alkylating agents (e.g., cyclophasphamide, melphalan,bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g., cisplatin(also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.),anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibioticagents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide,tenoposide, and camptothecins), or other cytotoxic agents such as ricin,diptheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin, saporin,pokeweed viral protein, ethidium bromide, glucocorticoid, anthrax toxinand others. See, e.g., U.S. Pat. No. 5,932,188.

The human IL-1α specific mAb can also be conjugated to a detectablelabel. Useful detectable labels in the present invention include biotinor streptavidin, magnetic beads, fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga,⁶⁸Ga, or ⁷²As), radioopaque substances such as metals for radioimaging,paramagnetic agents for magnetic resonance imaging, enzymes (e.g.,horseradish peroxidase, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters. Fluorescent markers may also be used andcan be detected using a photodetector to detect emitted illumination.Enzymatic labels are typically detected by providing the enzyme with asubstrate and detecting the reaction product produced by the action ofthe enzyme on the substrate, and colorimetric labels are detected bysimply visualizing the colored label.

The present invention also encompasses nucleic acid molecules encodingfully human mAbs specific for human IL-1α. Although the same nucleicacid molecule might encode both the heavy and light chains of a humanIL-1α-specific mAb, two different nucleic acid molecules, one encodingthe heavy chain and the other encoding the light chain might also beused. The amino acid sequences of three IgG1 mAbs specific for humanIL-1α are presented herein. See SEQ ID NOs: 1, 3, 5, 7, 9, and 11.Exemplary nucleic acid molecules encoding these amino acid sequences arealso described herein. See SEQ ID NOs: 2, 4, 6, 8, 10, and 12. Any othersuitable nucleic acid that encodes the amino acid sequences of the twodescribed IgG1 mAbs or other mAbs within the invention might also beused.

For production of mAbs, the nucleic acid molecules of the inventionmight be incorporated into an expression vector in an orientationwherein such nucleic acid molecules are operatively linked to expressioncontrol sequences such as transcriptional and translational controlsequences. Examples of expression vectors include vectors derived fromplasmids and vectors derived from viruses such as adenoviruses,adeno-associated viruses, and retroviruses. The nucleic acid moleculesencoding a light chain and a heavy chain might be incorporated into asingle vector or different vectors. The vectors of the invention mightalso include regulatory sequences such as promoters and/or enhancers(see, U.S. Pat. No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No.4,968,615), selectable markers, or sequences encoding affinity tags (forfacilitating purification) or a detectable label.

For production of mAbs, the vectors of the invention can be introducedinto a suitable host cell, e.g., a prokaryotic cell such as a bacteriaor, preferably, a eukaryotic cell such as mammalian, plant, or yeasthost cell. Examples of methods for introducing heterologouspolynucleotides into host cells include use of viral vectors,electroporation, encapsulation of the polynucleotide(s) in liposomes,dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion,Agrobacterium-mediated transformation, biolistic transformation, anddirect microinjection of the DNA into nuclei. Mammalian cell lines arepresently preferred for expression of mAbs from vectors. Examples ofmammalian host cells include Chinese hamster ovary (CHO) cells (e.g.,the DG44 CHO cell line), HeLa cells, baby hamster kidney (BHK) cells,African green monkey kidney cells (COS), human hepatocellular carcinomacells (e.g., Hep G2), NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells, and NIH-3T3 cells. The mAbs of the invention might alsobe expressed in transgenic animals or plants. See, e.g., U.S. Pat. Nos.5,827,690; 5,756,687; 5,750,172; 5,741,957; 6,046,037; and 5,959,177.

The invention provides a method for detecting a human IL-1α-expressingcell in a sample by contacting the cell with a human IL-1α-specific mAband detecting the mAb bound to the cell. The invention also provides amethod for killing a human IL-1α-expressing cell by contacting the cellwith a human IL-1α-specific mAb. Such killing can be accomplished bycomplement-mediated killing, Ab-dependent cell-mediated cytotoxicity, orAb-mediated delivery of a cytotoxin. The Abs described herein have alsobeen shown to be useful for other methods.

For example, MABp1 has been to reduce IL-1α induced ICAM1 and E-selectinexpression on endothelial cells. MABp1 has also been shown to be used inimmunoassays for detecting and quantifying IL-1α in a biological sample.

EXAMPLES Example 1 Cloning of Anti-hIL-1α IgG1 and Kappa Chains

Variable region heavy chain (V-HC) and variable region light chain(V-LC) sequences were gene synthesized using amino acid sequenceinformation provided in U.S. Pat. No. 5,959,085. V-HC was PCR amplifiedintroducing HindIII/ClaI sites upstream of the ATG start codon and aNheI site at the 3′ end. The human germline IgG1 constant region(including exons and introns) was PCR amplified modifying the two 5′triplets encoding for the first two amino acids Ala-Ser to an NheI site,and introducing a BamHI site at the 3′ end. The human germline IgG1constant region amino acid sequence corresponded to Swiss-Prot entryP01857, except for a K171Q and a V261L exchange. The V-HC and constantIgG1-HC sequence were ligated using the NheI site and cloned into pcDNA3using HindIII and BamHI sites.

>hIL-1a-IgG1-HC (SEQ ID NO: 1)MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAQTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIALEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >hIL-1a-IgG1-HC (SEQ ID NO: 2)atggagttcgggctgagttgggtgttcctggtggctctgctgcggggcgtgcagtgccaggtgcagctggtggagagtgggggtggcgtggtgcagcctggccggtctctgcgcctgtcttgcactgcctccggttttaccttttctatgtttggtgtgcactgggtgcgccaggctcccggcaagggactggaatgggtggccgccgtgagttacgacgggtccaacaaatattacgctgagagcgtgaaaggcagattcaccatcagcagagataattccaagaatattctgttcctgcagatggacagtctgagactggaggacactgctgtgtactactgcgctcgtggacgccctaaggtggtcatccccgcccccctggcacattggggccagggaactctggtgaccttttctagcgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtccacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcccagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccctggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcctt aagtccgggaaaataa

The V-LC was PCR amplified introducing HindIII/ClaI sites upstream ofthe ATG start codon and a BsiWI site at the 3′ end. The human constantKappa-LC sequence was PCR amplified introducing a 5′ BsiWI site encodingan additional Arg and the first amino acids Thr, and a BamHI site at the3′ end. The human constant Kappa-LC amino acid sequence corresponded toSwiss-Prot entry P01834. V-HC and constant Kappa-LC sequences wereligated using the BsiWI site and cloned into pcDNA3 using HindIII andBamHI sites.

>hIL-1a-K-LC [SEQ ID NO: 3]MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGGGTKVEHRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >hIL-1a-K-LC [SEQ ID NO: 4]atggacatgcgcgtgcccgcccagctgctggggctgctgctgctgtggttccctggatctaggtgcgacattcagatgacccagtcccccagctcagtgtcagcctccgtgggcgacagagtgacaatcacctgccgcgcctctcagggaatctctagttggctggcctggtaccagcagaagcctggaaaggcccccaagctgctgatctatgaagcctccaacctggagaccggcgtgccctctcgcttcagcggctcaggctcaggcagtgattttactctgaccatcagctccctgcagccagaggatttcgctacttactactgccagcagacctcttccttcctgctgtccttcgggggaggcacaaaggtggagcaccgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagttcaccggtgacaaagagcttcaacaggggag agtgttag

Example 2 Generation of NATHMAB-hIL-1a IgG1 and Kappa Chain

The complete sequence encoding the NATHMAB-hIL-1a/IgG1 heavy chain wasgene synthesized. The V-HC sequence corresponded to the amino acidsequence described in U.S. Pat. No. 5,959,085. The human constantIgG1-HC sequence corresponded to Swiss-Prot entry P01857. The nucleotidesequence was codon optimized for expression in CHO cells. A Kozacsequence (gccacc) was added upstream of the start ATG.

>NATHMAB-hIL-1A-IGG1-HC [SEQ ID NO: 5]MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >NATHMAB-hIL-1A-IGG1-HC [SEQ ID NO: 6]gccaccatggagtttggtctgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagctggtggagtctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatgtttggtgtgcattgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatattacgctgagtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctgaggctggaagatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggacagggaaccctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctacttctggaggcaccgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctgactagcggcgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctccagcagcctcggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagaaggtcgaacctaaatcttgcgataaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttcccccccaaacccaaagataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaagtcaaattcaactggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacatatagggtggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaaggccctgccagccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccctctcgggatgaactgaccaagaatcaggtgagcctgacatgtcttgtgaagggtttttatccctccgacattgccgtggagtgggagagcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggttccttcttcctctactctaagctgacagtggataagtctaggtggcagcaggggaatgtgttctcctgctctgtgatgcacgaggcactgcacaatcattatacacaaaagtctctgtctctgtctccaggaaagtaa

The complete sequence encoding the NATHMAB-hIL-1a/Kappa light chain wasgene synthesized. The V-LC sequence corresponded to the amino acidsequence described in U.S. Pat. No. 5,959,085. The human constantKappa-LC sequence corresponded to Swiss-Prot entry P01834. Thenucleotide sequence was codon optimized for expression in CHO cells. AKozac sequence (gccacc) was added upstream of ATG.

>NATHMAB-hIL-1A-K-LC [SEQ ID NO: 7]MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGGGTKVEHTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NATHMAB-hIL-1A-K-LC [SEQ ID NO: 8]gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtgatattcagatgacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccaggggatctctagttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcgaaacaggcgtgccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgaggattttgccacatattactgtcagcaaacttcttcttttctgctctcctttggtgggggaactaaggtggagcacacagtggccgcccccagcgtctttatcttccccccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataatttttaccctcgcgaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagcaagacagcaaggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgtacgcttgtgaggtgactcaccagggactgagtagccctgtgacaaaatctttcaataggg gagaatgctga

Example 3 Expression of NATHMAB-IL1-a (IgG1/k Subtype)

NATHMAB-IL-1α was expressed and purified using a transient transfectionmethod. Cell culture supernatant or protein G affinity purified Ab wassubjected to further analysis as described below. Human embryonic kidney(HEK) 293T cells were cultured in DMEM containing 10% FCS, andtransiently transfected using jetPEI reagent (Polyplus) according tomanufacturer's protocol. Cells were seeded on 10 cm dishes (3×10⁶ cellsper 10 cm dish) 24 h prior to transfection to reach approximately 50%density at the time point of transfection. 5 μg per dish ofpcDNA3-anti-hIL-1α-IgG1-HC and a 2-fold molar excess ofpcDNA3-anti-hIL-1α-Kappa were used for transfection. After recovery,medium was changed to DMEM containing 2% FCS (10 ml per dish) and Ab wascollected for 5 to 6 days. The supernatant was collected, filtered, pHadjusted to 7.5-8, and stored at 4° C. until further use.

Part of the supernatant (250 ml) was incubated with protein G sepharose(GE Healthcare) for 3 h at 4° C. on a rotation wheel. Then, the proteinG sepharose was loaded onto a gravity flow column and washed with PBS.Ab was eluted in 1 ml fractions using 100 mM glycine/150 mM NaCl into100 μl Tris (pH 8), followed by dialysis with PBS containing 10%glycerol. The total protein concentration of each fraction was measuredusing the BCA Protein Detection Kit (Pierce). Correct size of heavy andlight chains, and of the assembled native Ab was confirmed by SDS-PAGE.

Supernatant containing NATHMAB-hIL-1α purified Ab and Triton X-100 celllysates of producer HEK 293T cells were tested for antigen binding in aradioimmunoassay (RIA) using ¹²⁵I-hIL-1α. Binding was assayed byabsorption to protein G. All samples bound ¹²⁵I-hIL-1α with highestactivity in the eluate. Binding of purified NATHMAB-hIL-1α in aconcentration of 0.012% (half-max activity in RIA) to ¹²⁵I-hIL-1α wasused for measuring the affinity coefficient. The Ka of NATHMAB-hIL-1αunder these conditions was 3.03×10¹⁰ M⁻¹. Back calculation revealed anestimated concentration of approximately 30 μg/ml active anti-hIL-1α-IgGin the purified eluate.

Neutralizing activity of NATHMAB-hIL1α was tested in a bioassay usingthe murine EL4-6.1 subline which produces high levels of IL-2 whentreated with murine or human IL-1α (Zubler et al., J. Immunol.134:3662-3668, 1985). The indicated concentrations of NATHMAB-hIL-1α(eluate) were incubated for 30 min at 37° C. with various concentrationsof recombinant hIL-1α (eBioscience) in a final volume of 100 μl/well ina 96-well culture plate (flat bottomed). Each point was carried out intriplicate and in culture medium (DMEM, 5% FCS). To each well were added100 μl of a suspension of EL4-6.1 cells (5×10⁵ cells/ml) in culturemedium containing 0.2 μg/ml ionomycin. After incubation for 24 h at 37°C. in a 5% CO₂ incubator, cell free supernatants were harvested andassayed for IL-2 concentrations using a commercially available ELISA(R&D Systems). The results showed that NATHMAB-IL-1α effectivelyneutralized hIL-1α-induced IL-2 secretion by EL-4 cells.

To test for neutralization of membrane-bound hIL-1α, the same EL-4cell-based assay as described above was used with followingmodifications. Different concentrations of NATHMAB-hIL-1α (eluate) wereincubated with various numbers of human activated monocytes. Formonocyte preparation, PBMC were isolated from buffy coat usingFicoll-Paque centrifugation. Monocytes were allowed to adhere for 1.5 hat 37° C. in RPMI on plastic dishes. Non-adherent lymphocytes werewashed away to yield a nearly pure monocyte culture. Monocytes werecultured in RPMI containing Gln, Pyr, and 10% FCS for 24 h with LPS (1μg/ml) at 37° C. in a 5% CO₂ incubator. Cells were detached with PBS/2mM EDTA, carefully scraped from plates, and transferred into Falcontubes. Cells were washed twice with PBS, resuspended in PBS/1% PFA andfixed for 10 min at 20° C. Cells were washed with glycine buffer (150 mMglycine, 75 mM NaCl, pH 7.4), then with culture medium and counted. Theresults showed that NATHMAB-hIL-1α effectively neutralized IL-2secretion by EL-4 cells induced by membrane-bound hIL-1α. In anexperiment similar to that described above, NATHMAB-hIL-1α was testedfor neutralization of murine IL-1α. Indicated amounts of NATHMAB-hIL-1αsupernatant were incubated with recombinant human (h) or murine (m)IL-1α (eBioscience). The supernatant containing the Ab neutralizedhuman, but not murine, IL-1α.

Example 4 Ab-Mediated Killing of Cancer Cells

Human peripheral blood mononuclear cells (PBMC) isolated from the buffycoat by standard Ficoll Paque preparation were incubated in eitherRPMI-1640 CM or RPMI-1640-CM containing rhIL-2 (30 ng/ml, ebioscience)at 37° C. and 5% CO2 overnight and used as effector cells (E). THP1cells were used as the targets (T). The assay was carried out in 96-wellplates with each point in triplicate. After 1×10⁴ targets that wereincubated with different concentration of MABp1 for 15 mins, effectorcells were added in an ET ratio of 25:1 and 50:1 to 1×10⁴ targets andincubated for another 4 hours. 75 ul of assay volume were transferred toa new 96-well plate and cytotoxicity was assayed using the LDHcytotoxicity detection kit (Roche) according to manufacturer's protocol.% specific lysis=(mean experimental release−mean spontaneous releasewithout antibody)×100/(mean maximal release from targets−meanspontaneous release from targets) A. untreated PBMC were used aseffector cells. B. rhIL-2-treated PBMC were used as effector cells. Inboth cases, increasing concentrations (1.25 to 20 ug/ml) of MABp1resulted in increased target cell killing (up to about 90%) at both ETratios.

Example 5 Human Anti-IL1α Specific mAb Sequences

The complete sequence encoding for another human anti-hIL-1algG₁/Kappalight chain specific for human IL1α (MABp1) was synthesized andexpressed as described above. In the nucleic acids encoding the heavyand light chains, a Kozac sequence (gccacc) was added upstream of thestart ATG.

Heavy Chain

[SEQ ID NO: 9] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK[SEQ ID NO: 10] gccaccatggagtttggtctgtcctgggtgttcttggtggctctgctgaggggggtgcagtgccaggtccagctggtggagtctggtgggggagtggtgcagcctgggagatctctgcggctgtcttgcactgcctctggtttcactttctctatgtttggtgtgcattgggtcaggcaagcaccaggcaaaggactcgagtgggtcgcagctgtgagctatgacgggtctaacaaatattacgctgagtctgtcaagggtaggtttaccatcagccgggataattccaaaaatatcctgttcctgcaaatggactctctgaggctggaagatactgcagtctactattgtgcaagggggaggccaaaggtggtgatccccgctcccctcgctcactggggacagggaaccctggtgactttcagctctgctagcaccaagggccctagcgtgttcccattggctccttcctccaaatctacttctggaggcaccgccgccctgggatgtctcgtgaaagattattttcctgagcccgtcaccgtgagctggaacagcggcgccctgactagcggcgtgcacacctttcccgcagtgctgcaatctagcgggctgtactccctgagctctgtcgtgaccgtgccctccagcagcctcggaactcagacctacatctgcaatgtcaatcataaaccctctaataccaaagtcgataagagggtcgaacctaaatcttgcgataaaacccatacctgccccccttgcccagcacccgaactgctgggcggtccctctgtgtttctgttcccccccaaacccaaagataccctgatgatctctaggacccccgaggtcacttgtgtcgtggtggatgtgtcccacgaagatccagaagtcaaattcaactggtatgtggacggggtcgaagtgcacaacgcaaagaccaagcctagggaggaacagtataatagcacatatagggtggtcagcgtcctgaccgtcctgcatcaggactggctgaatggcaaagaatataagtgtaaagtgtccaacaaggccctgccagccccaatcgaaaagacaatctctaaagccaaggggcaaccccgggaacctcaggtctatacactgccaccctctcgggaggaaatgaccaagaatcaggtgagcctgacatgtcagtgaagggtttttatccctccgacattgccgtggagtgggagagcaatggacaaccagaaaataactacaaaaccacaccccctgtgctggactccgatggaccacttcctctactctaagctgacagtggataagtctaggtggcagcaggggaatgtgactcctgctctgtgatgcacgaggcactgcacaatcattatacacaaaagtctctgtct ctgtctccaggaaagtaa

Light Chain

[SEQ ID NO: 11] MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKWYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGGGTKVEHKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 12]gccaccatggacatgcgcgttcctgcccagctcctcggactgctgctgctttggttcccaggctcccggtgtgatattcagatgacacagtctccctcctccgtatctgcatccgtgggcgacagggtcacaatcacttgtagggccagccaggggatctctagttggctcgcatggtaccaacaaaagccaggtaaggctccgaaactgctcatttacgaagctagtaacctcgaaacaggcgtgccaagccggtttagcggctccggttccggttctgacttcaccctcactatttcctccctgcaacctgaggattttgccacatattactgtcagcaaacttcttcttttctgctctcctttggtggaggaactaaggtggagcacaagcggacagttgctgctcctagcgtctttatcttccctccaagcgatgaacagctgaagtcagggaccgccagcgtggtctgcctgctcaataatttttaccctcgcgaggctaaggtccaatggaaagtggataacgccctccagagcggtaactctcaggagtctgtcacagagcaagacagcaaggatagcacctattccctctccagcaccctgacactgtctaaggccgactacgagaaacacaaagtgtacgcttgtgaggtgactcaccagggactgagtagccctgtgacaaaatctttca ataggggagaatgctga

Example 6 MABp1 Binding Affinity

The binding affinity of purified MABp1 was determined using surfaceplasmon resonanace (SPR) on a BIAcore 2000 instrument (GE HealthSciences). A mouse monoclonal anti-human IgG (Fc) Ab was covalentlyimmobilized on the flow cells of a CM5 sensor chip using a human Abcapture kit and amine coupling kit (GE Health Sciences). Immobilizationlevels of 8000-14000 RU would typically be achieved. Afterimmobilization of the mouse anti-human IgG (Fc) capture Ab, threestart-up cycles with HBS-EP running buffer (GE Health Sciences) and twostart-up cycles with MABp1 were run to stabilize the CM5 surface and toremove any non-covalently bound Ab. For analysis, MABp1 Ab was dilutedinto HBS-EP running buffer to a final concentration of 1 μg/mL andimmobilized to 700 RU on one flow cell of the CM5 sensor chip.Carrier-free human IL-1A cytokine (eBioscience, #34-8019) was seriallydiluted in HBS-EP running buffer over a test range from 100 nM to 0.05nM. Flow rate was 30 μl/min. Dissociation data for each cytokinedilution was recorded for 15 minutes. The CM5 surface was regeneratedafter each cycle using a single injection of 3 M MgCl₂ for 25 seconds ata flow rate of 30 μl/min. BiaEvaluation software and a Langmuir bindingmodel was used to fit the data. The K_(D) for MABp1 was determined to beless than 2.0×10⁻¹⁰ M.

Example 7 MABp1 Inhibits Tumor Cell Invasion of a Basement MembraneMatrix

Matrigel (BD), a basement membrane matrix, was thawed at 4° C. overnightand the dilute (5 mg/ml to 1 mg/ml) in serum free cold cell culturemedia. 100 ul of the diluted matrigel was placed into the upper chambersof a 24-well transwell (Costar) and the transwell was incubated at 37°C. for at least 4 to 5 h for gelling. Tumor cells (MDA-MB-231 and THP-1)were harvested from tissue culture flasks by Trypsin/EDTA, washed withculture media, and resuspended in medium containing 1% FBS at a densityof 1×10⁶ cells/ml. The gelled matrigel was gently washed with warmedserum free-culture media, and 100 ul of the cell suspension was added ineach well. The lower chamber of the transwell was filled with 600 ul ofculture media, and the plates was incubated at 37° C. for 12 to 24 h.The cells that did not invade the matrigel were gently scraped off thetop of each transwell with a cotton swab. The transwells were thenremoved from the 24-well plates and stained with crystal violet afterfixing the invaded cells with 70% ethanol or methanol. The invaded cellswere counted under a light microscope. The percent of cells invading thematrigel was significantly inhibited in the presence of MABp1.

Example 8 MABp1 Blocks Increase in ICAM1 Expression in Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) (BD Biosciences) wereseeded to 24-well plates at 5×10⁵ per well in 1 mL of M-200 mediumsupplemented with low-serum growth supplement (Invitrogen). Cells wereallowed to settle for 3-4 hours. Medium was aspirated and a fresh 1 mLof M-200 was added per well. MABp1 was added directly to cells @ 4.26μg/mL, co-incubated for 15 minutes at room temperature, and thenrecombinant human IL-1α (rhIL1A, eBioscience) was added to a finalconcentration of 40 pg/mL. Positive control wells received the additionof IL-1α only. HUVEC cells in the absence of IL-1α or the absence ofMABp1 served as negative controls. After 17-20 hours incubation at 37°C., 5% CO₂, cells were lifted from the plates by a non-enzymatictreatment for 20 minutes using CellStripper reagent (Cellgro Mediatech)and then immediately assayed for CD54 (ICAM-1) expression using standardflow cytometry protocols. Staining buffer comprised Dulbecco's PBSsupplemented with 2% heat-inactivated fetal bovine serum. PE-conjugatedmouse anti-human CD54 (ICAM-1) mAb (eBioscience, clone HA58) or aPE-conjugated mouse IgG1k isotype control (eBiocience, #12-4714) wereused per manufacturer's instructions to stain HUVEC cells in a 100microliter staining volume for 20 minutes in the dark at roomtemperature. Two washes in staining buffer were subsequently performedand then samples were acquired on a FACSCalibur flow cytometer (BDBiosciences). Among several independent experiments (n=5) theupregulation of ICAM-1 adhesion molecules induced by rhIL1A on thesurface of HUVEC cells was neutralized by MABp1 to baseline levelsexhibited by the unstimulated HUVEC cells.

Example 9 MABp1 Blocks Increase in E-Selectin Expression in EndothelialCells

Similar to its effects on ICAM-1 induction, MABp1-mediatedneutralization of induction of CD62E (E-selectin) on HUVEC cells wasalso observed. This effect was most pronounced when HUVEC cells werestimulated not by soluble rhIL-1a but by membranous IL-1a anchored byglycosyl-phosphatidylinositol to the surface of DG44 CHO cells (GPI-IL1Acells). In this experiment, confluent cultures of HUVEC cells in 6-wellplates were co-cultured overnight with 5×10⁶ GPI-IL1A DG44 cells inM-200 medium, either alone, in the presence of 10 μg/mL MABp1, or in thepresence of 10 μg/mL D5 isotype control Ab. After 17-20 hours, HUVECmonolayers were washed extensively with Dulbecco's PBS and then liftedby non-enzymatic treatment for 20 minutes with CellStripper reagent(Cellgro Mediatech) and then immediately assayed for CD62E (E-selectin)expression using standard flow-cytometry protocols. Staining buffercomprised Dulbecco's PBS supplemented with 2% heat-inactivated fetalbovine serum. PE-conjugated mouse anti-human CD62E mAb (eBioscience,clone P2H3) or a PE-conjugated mouse IgG1k isotype control (eBiocience,clone P3) were used per manufacturer's instructions to stain HUVEC cellsin a 100 microliter staining volume for 20 minutes in the dark at roomtemperature. Two washes in staining buffer were subsequently performedand then samples were acquired on a FACSCalibur flow cytometer (BDBiosciences). Upregulated E-selectin expression on the surface of HUVECcells induced by membranous GPI-IL-1a was neutralized by MABp1 tobaseline levels exhibited by unstimulated HUVEC cells.

Example 10 MRC-5 Bioassay for MABp1 Potency (Neutralization of rhIL1A)

The MRC-5 cell line, derived from fetal human lung fibroblasts, wasobtained from the ATCC collection (CCL-171). The IL-1 neutralizingpotency of MABp1 was assayed by measuring IL-1A induced release of IL-6from MRC-5 cells. MRC-5 cells were seeded at 5×10³ per well to a 96-wellplate in 100 microliters of DMEM complete medium. Cells were culturedovernight at 37° C. in a humidified 5% CO₂ incubator. Confluent MRC-5cells were subsequently cultured another 24 hours with 20 pg/mL ofrecombinant human IL-1A (rhIL1A, eBioscience) either alone or in thepresence of increasing concentrations of MABp1. Negative control cellswere not stimulated with rhIL1A. After the 24 hours, supernatants werecollected and assayed for IL-6 release using and IL-6 ELISA kit fromeBioscience. The IC₅₀, or concentration of MABp1 required to inhibit 50%of the maximal IL-6 release, was in the range of 0.001-0.01 μg/mL.

Example 11 MABp1 Identifies IL-1a+ Cells

One hundred microliters of sodium heparin anti-coagulated whole bloodwas aliquoted to polystyrene FACS tubes. Samples were incubated at roomtemperature for 15 minutes with 1 mg of human IgG (protein-A purified)plus 2 ml of heat-inactivated fetal bovine serum to block Fc receptors.Primary Abs were then added to the sample: Either 1 mg of Alexa-488labeled MABp1, 1 mg of FITC-labeled monoclonal anti-membrane human IL1AAb (FAB200F, R&D Systems), or 1 mg of a murine isotype control (IC002F,R&D Systems). Primary Abs were incubated with sample for 30 minutes atroom temperature in the dark. Sample erythrocytes were then lysed (BDBiosciences PharmLyse solution) at room temperature for 15 minutes,centrifuged at 300×g for 5 minutes, and aspirated. Sample pellets werewashed three times with 1 mL Hank's balanced salt solution (HBSS)containing 2% heat-inactivated fetal bovine serum. Sample wasresuspended in 0.3 mL HBSS+2% FBS and data was acquired on a FACSCaliburflow cytometer and analyzed using CellQuest software. Flow cytometricanalysis of human PBMC using MABp1 showed that only 0.2% of PBMC werepositive for IL-1α.

Example 12 MABp1 for Detecting and Tracking Infections and Inflammation

Flow cytometric analysis (as in Example 11) of human PBMC using MABp1showed a 3.6-fold increase in the percent of PBMC positive for IL-1α⁺ ina subject with a sub-clinical infection compared to a normal control.Similarly, in a subject with an inflamed wisdom tooth, an increase inthe percent of PBMC positive for IL-1α⁺. A substantial decrease in thenumber of IL-1α⁺ PBMC was observed from 14 to 45 days after removal ofthe wisdom tooth.

Example 13 Immunoassay for Detecting and/or Quantifying IL-1α

In general, very low levels of IL-1α are present in the plasma of humansubjects. Because these levels are often beyond the detection thresholdof conventional immunoassays, an ELISA with improved sensitivity wasdeveloped. In this ELISA, exogenous anti-IL-1α Ab (e.g., MABp1) can beadded to a biological sample being tested (e.g., human plasma) underconditions that allow the Ab to bind IL-1α in the sample. Because, itwas observed that almost all IL-1α in human plasma samples existsalready bound to endogenous anti-IL-1α Ab, the latter step can often beomitted. The sample with IL-1a-Ab complexes is then applied to a filter(Amicon centrifugal device) with a molecular weight cutoff of about 100kDa to separate the IL-1α-Ab complexes from molecules in the sample lessthan the molecular weight cutoff. In one experiment, this resulted in a50-fold concentration. The processed sample (and dilutions thereof) wasthen added to wells of a microtiter plate coated with an anti-human IgGcapture Ab (2 ug/ml mouse anti-human IgG, Fc-specific, Southern Biotechproduct code #9042-01). After allowing time to bind the IL-1α-Abcomplexes in the sample, the wells were washed to remove non-bindingmaterial. A labeled anti-human IL-1α secondary Ab was then added to thewells (0.2 ug/ml biotin-conjugated monoclonal mouse anti-human IL-1A Ab,clone CRM6, eBioscience catalog #13-7017). After allowing time to bindthe IL-1α in the wells, the plate was washed and the amount of labeledanti-human IL-1α in each well was quantified as an indication of theconcentration of IL-1α in the sample being tested.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of killing a human cell expressing humaninterleukin-1 alpha (IL-1α), the method comprising the step ofcontacting the cell with a purified human IgG1 monoclonal antibody (mAb)that specifically binds to human IL-1α, wherein the mAb is conjugated toa cytotoxin.
 2. The method of claim 1, wherein the human IgG1 mAbcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO.9 and a light chain comprising the amino acid sequence of SEQ ID NO:11.3. The method of claim 1, wherein the human cell is a cancer cell.
 4. Amethod of killing a human cell expressing human IL-1α, the methodcomprising the step of contacting the cell with a purified human IgG1mAb that specifically binds to human IL-1α, wherein the step ofcontacting the cell with a purified human IgG1 mAb is performed byadding the mAb to a mixture comprising the human cell and humanperipheral blood mononuclear cells.
 5. The method of claim 4, whereinthe human IgG1 mAb comprises a heavy chain comprising the amino acidsequence of SEQ ID NO. 9 and a light chain comprising the amino acidsequence of SEQ ID NO:11.